U.S. patent application number 15/897896 was filed with the patent office on 2018-08-23 for novel bioactive compound obtained from oil palm base materials.
This patent application is currently assigned to Malaysian Palm Oil Board. The applicant listed for this patent is Malaysian Palm Oil Board. Invention is credited to Che Anishas Che Idris, Mohd Jamil Elias, Soon Sen Leow, Jabariah Md. Ali, Mohd Sofian Mohamad Ideris, Ravigadevi Sambanthamurthi, Syed Fairus Syed Abu Bakar, Yew Ai Tan, Wan Saridah Wan Omar.
Application Number | 20180236022 15/897896 |
Document ID | / |
Family ID | 49997934 |
Filed Date | 2018-08-23 |
United States Patent
Application |
20180236022 |
Kind Code |
A1 |
Sambanthamurthi; Ravigadevi ;
et al. |
August 23, 2018 |
NOVEL BIOACTIVE COMPOUND OBTAINED FROM OIL PALM BASE MATERIALS
Abstract
This disclosure is directed to a novel bioactive compound
obtained from oil palm based materials and compositions containing
said bioactive compound. The bioactive compound obtained in
accordance with the present invention has a molecular mass of 482.
The bioactive compound also has potent HIV reverse transcriptase
activity and antioxidant activity.
Inventors: |
Sambanthamurthi; Ravigadevi;
(Kajang, MY) ; Tan; Yew Ai; (Kajang, MY) ;
Syed Abu Bakar; Syed Fairus; (Kajang, MY) ; Che
Idris; Che Anishas; (Kajang, MY) ; Leow; Soon
Sen; (Kajang, MY) ; Elias; Mohd Jamil;
(Kajang, MY) ; Wan Omar; Wan Saridah; (Kajang,
MY) ; Mohamad Ideris; Mohd Sofian; (Kajang, MY)
; Md. Ali; Jabariah; (Kajang, MY) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Malaysian Palm Oil Board |
Kajang |
|
MY |
|
|
Assignee: |
Malaysian Palm Oil Board
KAJANG
MY
|
Family ID: |
49997934 |
Appl. No.: |
15/897896 |
Filed: |
February 15, 2018 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
14417741 |
Jan 27, 2015 |
9919020 |
|
|
PCT/MY2013/000135 |
Jul 26, 2013 |
|
|
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15897896 |
|
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 36/889 20130101;
A61K 2236/00 20130101; A61K 31/05 20130101; A61P 31/12
20180101 |
International
Class: |
A61K 36/889 20060101
A61K036/889; A61K 31/05 20060101 A61K031/05 |
Foreign Application Data
Date |
Code |
Application Number |
Jul 27, 2012 |
MY |
PI2012700505 |
Claims
1. A composition comprising a bioactive compound obtained from oil
palm based materials, wherein the molecular weight of said compound
is 482.
2. The composition as claimed in claim 1 wherein the bioactive
compound is extracted from palm oil based wastes.
3. The composition as claimed in claim 2 wherein the bioactive
compound is extracted from oil palm vegetation liquor.
4. The composition as claimed in claim 1 wherein the compound is
extracted from palm oil mill effluent.
5. Use of a compound obtained from oil palm based materials as the
active ingredient for the preparation of a composition useful for
providing bioactive properties. whereby the molecular weight of
said compound is 482.
6. The use according to claim 5, wherein the compound is obtained
from palm oil based wastes.
7. The use according to claim 5, wherein the compound is obtained
from oil palm vegetation liquor.
8. The use according to claim 5, wherein the compound is obtained
from palm oil mill effluent.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. application Ser.
No. 14/417,741, filed Jan. 27, 2015, which is a national stage of
PCT/MY2013/000135, filed Jul. 26, 2013, which claims benefit of
Malaysian App. No. PI 2012700505, filed Jul. 27, 2012; all of which
are incorporated herein by reference in their entireties.
FIELD OF INVENTION
[0002] The invention generally relates to bioactive compounds and
more particularly to a phenolic compound obtained from plants and
plant-based material, with said compound exhibiting highly
significant bioactive properties.
BACKGROUND OF INVENTION
[0003] The demand for bioactive compounds is expected to increase
dramatically with the increase in world population and thus the
need for various industrial and pharmaceutical uses, e.g in the use
of manufacturing medical remedies such as anti-viral drugs
particularly in the event of a pandemic outbreak.
[0004] At present a great majority of bioactive compounds are
generally found in substantially low concentrations. In addition,
the process or method of extraction is expensive. Further, the
scarce availability of bioactive compounds has hampered the
potential production of medicaments, and thus stresses the need for
other abundant sources. Accordingly, it would be desirable to
explore other low cost and abundant sources for bioactive compounds
in order to aid in fulfilling the surging global demand.
[0005] The present application focuses on realizing the value and
potential of the vegetation liquor and oil palm based materials
from palm oil milling and palm oil mill effluent (POME) as a source
of bioactive compounds.
[0006] The present invention discloses oil palm based materials
including the vegetation liquor of palm oil milling as an abundant
source of bioactive compounds.
SUMMARY OF INVENTION
[0007] In one aspect there is provided a composition comprising a
bioactive compound obtained from oil palm based materials, wherein
the molecular weight of said phenolic compound is 482.
[0008] In another aspect there is provided the use of a compound
obtained from oil palm based materials as the active ingredient for
the preparation of a composition useful for providing bioactive
properties, whereby the molecular weight of said compound is
482.
BRIEF DESCRIPTION OF DRAWINGS
[0009] Some figures contain color representations or entities in
order to elucidate the results of experiments for the purpose of
the present invention.
[0010] FIG. 1 shows the results obtained based on DPPH Scavenging
Assay with respect to the compound in accordance with a preferred
embodiment of the claimed invention; and
[0011] FIG. 2 shows the results obtained based on Reverse
Transcriptase Assay with respect to the compound in accordance with
a preferred embodiment of the claimed invention.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0012] The disclosed description and examples are directed to a
bioactive compound, composition and method thereof, whereby the
bioactive compound with molecular weight of 482 is extracted from
oil palm based materials.
[0013] The biologically active extracts of palm vegetation liquor
useful in this invention are those obtained from the vegetation
liquor of the palm oil milling process according to various
conventional suitable means and processes.
[0014] Although the extract may contain a variety of compounds
including phenolic compounds, fruit acids, fruit sugars and
glycerol, starch, cellulose and hemicellulose, for purposes of
standardization the concentrations of the extracts used were
measured in terms of phenolic content i.e gallic acid
equivalent.
[0015] Embodiments of the present invention are directed to a
composition comprising a bioactive compound and other major
phenolic compounds obtained from any part of the oil palm, oil palm
based materials including vegetation liquor of palm oil processing
and palm oil mill effluent. The composition of the present
invention can be prepared based on available or standard methods.
It is expected that the preparation is safe and said composition is
suitable for use in, but not limiting to, daily consumption
including dietary supplements, nutraceuticals, and for health
promoting purposes. It is further noted that the bioactive compound
and its derivatives obtained based on the preferred embodiments of
the present invention are suspected to exhibit antiviral and
antimicrobial effects.
[0016] It would be apparent to a person skilled in the art that the
raw extracts obtained from any part of the oil palm, or oil palm
based materials, including the vegetation liquor from palm oil
milling and palm oil mill effluent for the purpose of the present
invention may contain various other phenolic compounds in addition
to the novel primary marker bioactive compound.
[0017] The extracts obtained from oil palm based materials and more
particularly for this disclosure, the vegetation liquor from palm
oil milling and palm oil mill effluent when subjected to isolation
and purification stages in accordance with the method of the
present invention are found to contain a novel bioactive compound
or their derivatives.
[0018] The present invention extends, therefore to a novel
bioactive compound; and method thereof, whereby the primary steps
of said method are pre-treatment of raw extracts obtained from any
part of the oil palm, the vegetation liquor from palm oil milling
and palm oil mill effluents. This embodiment encompasses isolation
of substantially purified bioactive compound having molecular
weight of 482. It should be noted that an "isolated or purified"
bioactive compound or biologically active portion thereof, is
substantially free of other cellular materials or other components
or substantially free of chemical precursors or other
chemicals.
[0019] The following examples serve to merely explain different
methods of preparing the bioactive compound and related compounds
and should not be construed to confine the scope of claims. In
accordance with a preferred embodiment, the extracts obtained from
the oil palm based materials, may be subjected to a pretreatment
process. Such process may be performed as portrayed in EXAMPLE 1
below.
EXAMPLE 1
Pretreatment of Extracts
[0020] The first step of the method for preparation of the
composition containing the bioactive compound is pre-treatment of
the raw extracts to obtain pre-concentrated or partially purified
extracts. This may be performed with low stringent conditions of
subjecting the extracts to a flash chromatography or the likes, or
alternatively, subjecting said extracts to ethanol precipitation,
prior to separation by high performance liquid chromatography. An
example of such method is given by way of reference below and
should not be construed as limiting the scope of the claims.
[0021] The main steps involved for the first approach is loading a
"sep-pak" type column, removing impurities, eluting said extracts
with methanol or ethanol and subjecting said extracts for
concentration stage in a rotary evaporator. The second approach
comprises the steps of adding an amount of extract to three volumes
of cola ethanol (EtoH), storing said mixture overnight at a
preferred temperature of -20.degree. C., centrifuging at 1500 Xg
for at least 15 minutes, dissolving the precipitate obtained from
the previous step with a suitable amount of distilled water and
concentrating by rotary evaporator at 50.degree. C. to obtain the
preferred final value of 3 ml. It should be mentioned, that these
steps for both approaches might be substituted with alternative
steps of standard procedures known in the art to achieve a similar
objective.
[0022] The next imperative step of the method for the preparation
of the composition as disclosed involves the isolation and
purification, of the phenolic compound from the partially purified
or pre-treated extracts. This can be carried out with the
conventional high performance liquid chromatography (HPLC) based on
low stringent conditions or parameters. An example of such method
is given by way of reference below and should not be construed as
limiting the scope of the claims.
EXAMPLE 2
Isolation and Purification of Phenolic Compounds from Oil Palm
Based Materials
[0023] An econosil C18 5 .mu.m particle size, with the preferred
column length of 25 cm.times.10 mm id, flow rate of 3 ml per minute
was prepared. The preferred mobile phase gradient may comprise two
solvents, with one solvent consisting of 0.1% trifluoroacetic acid
(TFA) with an amount of water and another solvent consisting of
10/90 of 0-1% TFA/acetonitrile (ACN) v/v. In this study, the
injection column was 1 ml and readings were taken at 280 nm. The
mixture is subjected to isolation by HPLC and it is observed that
there are several peaks, at least one peak indicating the elution
of the compound within 30 to 35 minutes. Further details of the
peaks will be described in the next example, which is the peak
isolation segment.
[0024] It would be understood that the choice of columns and
parameters for HPLC may vary however to obtain a similar result of
elution time as described. Eluted fraction may be suitably
collected and provided in powder or liquid form for use in further
analysis.
EXAMPLE 3
Peak Isolation
[0025] The HPLC chromatogram obtained from the injection of
concentrated sample was observed. Identification of peak fractions
was based on retention time. From the chromatogram, there were
several peaks observed, wherein each of the peak fractions was
subjected to structural and chemical identification.
EXAMPLE 4
Molecular Weights of Peaks
[0026] The molecular weights of the six major peaks collected were
obtained using standard Liquid Chromatography--Mass Spectrometry
(LC-MS). From the results, it was observed that peak 6 has a
molecular mass of 482.
EXAMPLE 5
Profiling--Free Radical Scavenging
[0027] Fractions 1 to 6 were further analysed for their properties
in free radical scavenging. The free radical form of DPPH* is
purple in colour and absorbs maximally at a wavelength of 515 nm.
Antioxidants such as certain phenolic compounds are able to
scavenge the free radicals of DPPH* resulting in a decrease in
intensity of the purple colour, which can be measured
spectrophotometrically.
[0028] Free Radical Scavenging Assay
[0029] Stock solution of DPPH* was diluted to 0.025 mg/ml with
water to give a final solution in 50% methanol. Gallic acid was
prepared at the concentration of 300 ppm (300 .mu.g/ml). Substances
to be tested were prepared in water to give a concentration of 300
ppm GAE.
[0030] To 975 .mu.l of DPPH* solution in a cuvette, 25 .mu.l of
sample was added. Absorbance at the wavelength of 515 nm was
monitored spectrophotometrically at 0.1 min intervals for 2 min.
The control was treated in the same manner except that the sample
was replaced with water. Blank contained 50% of methanol in place
of DPPH* and water in place of sample. Values for the blank were
subtracted from the test values.
[0031] Concentration of DPPH* at any particular absorbance was
calculated from the DPPH* standard curve using the formula,
Y=aX+b
[0032] Where, Y=absorbance (at 515 nm) [0033] X=concentration of
DPPH* (.mu.g/mL) [0034] a=Linear regression coefficient [0035]
b=y-intercept
[0036] Since the standard curve passes through the origin,
therefore b=0
[0037] Rearranging the formula,
X = Y a . ( 3 ) ##EQU00001##
[0038] The percentage of DPPH* remaining (% DPPH*.sub.rem) was
calculated using the formula,
% DPPH rem * = [ DPPH ] 0 - [ DPPH 0 ] t [ DPPH 0 ] 0 .times. 100 %
( 4 ) ##EQU00002##
[0039] Where,
[0040] [DPPH*].sub.t=concentration of DPPH* at t time
(.mu.g/ml)
[0041] [DPPH*].sub.0=initial concentration of DPPH* (.mu.g/ml)
[0042] Percentages of DPPH* remaining against time: were: plotted
and the graph obtained is shown in FIG. 1. According to the results
obtained, peak 6 exhibited the strongest DPPH free-radical
scavenging activity owing to its lowest absorption compound to
other peaks.
[0043] Further chemical analysis on determining the properties of
the phenolic compound based on the peaks as obtained in accordance
with the method of the present invention may be carried out based
on conventional or standard procedures known in the art.
EXAMPLE 6
Reverse Transcriptase Assay: Inhibitor Determination
[0044] Fractions of 1 to 6 were further analyzed for protease and
HIV reverse transcriptase inhibiting properties. In accordance with
one embodiment of the present invention, the purified fraction of
peak 6 of the disclosed invention has shown potent inhibitory
action against both HIV protease and reverse transcriptase.
Compound of molecular weight 482 corresponds to peak 6.
[0045] The preferred assay system for analyzing the human
immunodeficiency virus (HIV) replication activity in associated in
accordance to the present invention is the Reverse Transcriptase
system. According to studies in the relevant field, the viral
activity can foe determined by way of a Reverse Transcriptase
Assay. Inhibition of reverse transcriptase is thus indicative of
anti-viral and more specifically anti-HIV activity when HIV reverse
trancriptase is used in the assay.
[0046] Commercial Reverse Transcriptase Assay Kits were used to
confirm the anti-HIV properties of the fractions.
[0047] Calometric Roche.TM. Reverse Transcriptase Assay
[0048] This is a colorimetric enzyme immunoassay for the
quantitative determination of retroviral reverse transcriptase
activity by incorporation of dioxigenein- and biotin-labeled dUTP
into DNA.
[0049] The Calometric Roche.TM. Reverse Transcriptase Assay, takes
advantage of the ability of reverse transcriptase to synthesize
DNA, starting from the template/primer hybrid poly (A).times.oligo
(dT)15. Digoxigenin- and biotin-labeled nucleotides in an optimized
ratio are incorporated into one and the same DNA molecule, which is
freshly synthesized by the RT. The detection and quantification of
synthesized DNA as a parameter for RT activity follows a sandwich
ELISA protocol: Biotin-labeled DNA binds to the surface of
microtiter plate (MTP) modules that have been precoated with
streptavidin. In the next step, an antibody to digoxigenin,
conjugated to peroxidase (anti-DIG-POD), binds to the
digoxigenin-labeled DNA. In the final step, the peroxidase
substrate ABTS is added. The peroxidase enzyme catalyzes the
cleavage of the substrate, producing a colored reaction product.
The absorbance of the samples can be determined using a microtiter
plate (ELISA) reader and is directly correlated to the level of RT
activity in the sample.
[0050] Accordingly, the fraction was prepared at various
concentrations in both the dried and aqueous form. The four
fractions from flash chromatography were also prepared with varying
concentrations. It is observed that peak 6 exhibited significant
and the highest inhibitory action, as seen in FIG 2.
[0051] The novel compound of the claimed invention may be prepared
for use in a pharmaceutically effective or nutraceutically
effective amount, solely on its own or in combination with other
agents or compounds deemed appropriate by a person skilled in the
art. Further, compositions nay be prepared in a manner, and in a
form/amount as is conveniently practised.
[0052] Those skilled in the art will appreciate that the invention
described herein is susceptible to variations and modifications
other than those specifically described. It is to be understood
that the invention includes all such variations and modifications.
The invention also includes all of the steps, features,
compositions and compounds referred to or indicated in this
specification, individually or collectively, and any and all
combinations of any two or more of said steps or features.
* * * * *